PROJECTSUMMARY AmajorgoaloftheBRAINinitiativeistounderstandneuronalconnectivityandplasticityinthecontextof animalbehavior.Thefunctionsandconnectivityofneuronsareestablishedandmanifestedbytheirconstituent proteins.Monitoringtheorganizationofindividualproteinsinspecificneuronalsubtypesinbehavinganimals maythereforeprovideanimportantreadoutofcellularandcircuitpropertiesunderlyinganimalbehavior. However,itremainschallengingtovisualizeendogenoussynapticproteinorganizationinindividualneuronsin livinganimals.Moststudiesrelyontheoverexpressionoffluorescentlytaggedproteinsofinterest.Protein overexpressioncanalterproteinstoichiometry,trafficking,subcellularlocalization,andcellsignaling,ultimately affectingcellularandcircuitfunctions.Although?knock-in?strategiescaninprinciplebypassproblems associatedwithproteinoverexpression,theyresultinglobalexpressionofthelabeledprotein,leadingtohigh fluorescencebackgroundandalackofcell-specificcontrast.Otheralternativelabelingmethodsforvisualizing endogenousproteins,suchastheintracellularexpressionoffluorescentlytaggedintrabodiesandCRISPR- mediatedgeneediting,alsohavetheirownlimitations,includingpotentialoff-targeteffects. Tosolvetheaboveproblems,werecentlydevelopedanovelgeneticstrategycalledendogenouslabelingvia exonduplication(ENABLED).WehaveusedthismethodtolabelthecriticalpostsynapticmarkerproteinPSD- 95withtheyellowfluorescentproteinmVenusinallneurons,inasparsesubsetofneurons,orinspecific neuronalsubtypes.UnliketheconventionalapproachtovisualizingPSD-95viaoverexpression,ourstrategy doesnotresultinalteredneuronalfunctions,and,forthefirsttime,allowsforthemonitoringofPSD-95at endogenouslevelsinindividualneuronsinlivingmice.Despitetheseadvantages,theENABLEDstrategycan befurtheroptimizedtobroadenitsapplicabilityandtoenhanceitssensitivity.Furthermore,tocomprehensively examineneuronalfunctionsandconnectivity,additionalsynapticproteinswillneedtobelabeledatboththe presynapticandpostsynapticsides.Here,werequestfundstooptimizetheENABLEDstrategyanduseitto label12additionalcriticalsynapticproteinsinmice.WewillalsogenerateENABLEDmiceinwhichthe synapticproteinscanbelabeledusingdifferentcolorsforsimultaneousimaging.Thereagentswegeneratewill bemadeavailabletotheneurosciencecommunitytoprovideresearcherswithanunprecedentedabilityto monitorsynapticconnectivityandplasticityunderphysiologicalconditionsinbehavinganimals.